Such a microscope and/or microscopy method is known from, by way of example, the publication C. Müller and J. Enderlein, Physical Review Letters, 104, 198101 (2010), or EP 2317362 A1, which also lists further aspects of the prior art.
This approach achieves an increase in location accuracy by imaging a spot on a detection plane in a diffraction-limited manner. The diffraction-limited imaging process images a point spot as an Airy disk. This diffraction spot is detected in the detection plane in such a manner that its structure can be resolved. Consequently, an oversampling is realized at the detector with respect to the imaging power of the microscope. The shape of the Airy disk is resolved in the imaging of a point spot. With a suitable evaluation of the diffraction structure—which is detailed in the documents named (the disclosure of which in this regard is hereby cited in its entirety in this application) an increase in resolution by a factor of 2 beyond the diffraction limit is achieved.
However, it is unavoidable in this case of the detector, that it is necessary to capture a single image with multiple times more image information for each point on the sample that is scanned in this way, compared to a conventional laser scanning microscope (shortened to “LSM” below). If the structure of the single image of the spot is detected, by way of example, with 16 pixels, not only is the volume of data per spot 16-times higher, but also a single pixel contains, on average, only 1/16 of the radiation intensity which would fall on the detector of an LSM in a conventional pinhole detection. Because the radiation intensity is, of course, not evenly distributed across the structure of the single image—for example the Airy disk—in reality, even less—and particularly significantly less—radiation intensity arrives at the edge of this structure than the average value of 1/n for n pixels.
Consequently, the problem exists of being able to detect quantities of radiation at the detector at high resolution. Conventional CCD arrays that are typically used in microscopy do not achieve sufficient signal-to-noise ratios, such that even a prolongation of the duration for the image capture, which would already be disadvantageous in application per se, would not provide further assistance. APD arrays also suffer from excessively high dark noise, such that a prolongation of the measurement duration would result here as well in an insufficient signal/noise ratio. The same is true for CMOS detectors, which are also disadvantageous with respect to the size of the detector element because the diffraction-limited single image of the spot would fall on too few pixels. PMT arrays suffer from similar constructed space problems. The pixels in this case are likewise too large. The constructed space problems are particularly a result of the fact that an implementation of a microscope for high resolution can only be realized, as far as the effort required for development and the distribution of the device are concerned, if it is possible to integrate the same into existing LSM constructions. However, specific sizes of the single images are pre-specified in this case. As a result, a detector with a larger surface area could only be installed if a lens were additionally configured that would enlarge the image once more to a significant degree—i.e. several orders of magnitude. Such a lens is very complicated to design in cases where one wishes to obtain the diffraction-limited structure without further imaging errors.
Other methods are known in the prior art for high resolution which avoid the problems listed above that occur during detection. By way of example, a method is mentioned in EP 1157297 B1, whereby non-linear processes are exploited using structured illumination. A structured illumination is positioned over the sample in multiple rotary and point positions, and the sample is imaged on a wide-field detector in these different states in which the limitations listed above are not present.
A method which also achieves high resolution without the detector limitations listed above (i.e. a resolution of a sample image beyond the diffraction limit) is known from WO 2006127692 and DE 102006021317. This method, abbreviated as PALM, uses a marking substance which can be activated by means of an optical excitation signal. Only in the activated state can the marking substance be stimulated to release certain fluorescence radiation by means of excitation light. Molecules which are not activated do not emit fluorescent radiation, even after illumination with excitation light. The excitation light therefore switches the activation substance into a state in which it can be stimulated to fluoresce. Therefore, this is generally termed a switching signal. The same is then applied in such a manner that at least a certain fraction of the activated marking molecules are spaced apart from neighboring similarly-activated marking molecules in such a manner that the activated marking molecules are separated on the scale of the optical resolution of the microscope, or may be separated subsequently. This is termed isolation of the activated molecules. It is simple, in the case of these isolated molecules, to determine the center of their radiation distribution which is limited by the resolution, and therefore to calculate the location of the molecules with a higher precision than the optical imaging actually allows. To image the entire sample, the PALM method takes advantage of the fact that the probability of a marking molecule being activated by the switching signal at a given intensity of the switching signal is the same for all of the marking molecules. The intensity of the switching signal is therefore applied in such a manner that the desired isolation results. This method step is repeated until the greatest possible number of marking molecules have been excited [at least] one time within a fraction that has been excited to fluorescence.